Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.Limbs of adult urodele exhibit a remarkable ability to restore missing parts when they are accidentally lost and have provided investigators with an ideal experimental model to study the mechanism of the complete restoration of original pattern (1, 2). Generally, limb regeneration proceeds through five steps: (i) formation of wound epidermis, (ii) dedifferentiation of cells under the wound epidermis, (iii) formation of blastema, (iv) growth and differentiation of the blastema, and (v) pattern reformation. Dedifferentiation and blastema formation are unique features of urodele limbs and are of prime importance in the initial phase of regeneration (1, 2). The breakdown of interstitial connective tissues, cartilages, bones, and muscles under the wound epidermis seems to be a trigger of the dedifferentiation of liberated cells, because these cells start to lose the morphologic characteristics of their differentiated state concomitantly with the tissue demolition (2).It has been generally accepted that extracellular matrix (ECM) molecules rapidly turn over during processes involving tissue remodeling, such as wound healing, metamorphosis, and regeneration (2-4). ECM is thought to stabilize the differentiated state of cells and support the expression of their normal tissue-specific phenotypes (5, 6). Consequently, the degradation of the ECM destabilizes the differentiated state and would b...